Loret E P, Mansuelle P, Rochat H, Granier C
Laboratoire de Biochimie, CNRS UA 1179, INSERM U 172, Faculté de Médecine secteur nord, Marseille, France.
Biochemistry. 1990 Feb 13;29(6):1492-501. doi: 10.1021/bi00458a021.
Two scorpion neurotoxins active only on insects, the insect toxins AaH IT1 and AaH IT2, were purified from the venom of scorpions Androctonus australis Hector collected in Tozeur (Tunisia) and characterized. AaH IT2 was sequenced and found to differ in four amino acid positions from AaH IT, the single previously sequenced insect toxin [Darbon, H., Zlotkin, E., Kopeyan, C., Van Rietschoten, J., & Rochat, H. (1982) Int. J. Pept. Protein Res. 20, 320-330] which possessed an equal potential for paralyzing fly larvae. The basic amino acid residues of AaH IT1, which differs from AaH IT by one amino acid residue, were selectively chemically modified. Six derivatives were characterized. Their toxicity toward fly larvae and cockroach was determined, and their affinity for the AaH IT1 synaptosomal receptor from cockroach nerve cord was measured. Modification of His-30, Lys-34, and Arg-60 showed no significant effect on biological activity. However, the modification of Lys-28 or Lys-51 demonstrated that these two amino acids are important for toxicity. Furthermore, simultaneous modifications of both Lys-28 and Lys-51 led to a cumulative decrease in biological activity. AaH IT1 and AaH IT2 show similar CD spectra. The secondary structures content of AaH IT2 was estimated from circular dichroism data. Results showed that this class of toxin should possess an additional alpha-helical region and a beta-sheet strand, not found in toxins active on mammals. Attempts to localize these secondary structural features in the amino acid sequence of AaH IT2 indicated that these two regions would be located within the last 20 C-terminal amino acid residues. From these studies on secondary structures, it is possible to consider that toxins active on insects are more structurally constrained than those active on mammals; a decreased molecular flexibility may be, at least partially, responsible for the observed specificity of these toxins for the insect sodium channel. Furthermore, the two alpha-helices found in insect toxins enclosed the two conserved Lys-28 and Lys-51 and might thus be implicated in the toxic site of insect toxins.
从采自突尼斯托泽尔的澳毒疣蛛（Androctonus australis Hector）毒液中纯化并鉴定出了两种仅对昆虫有活性的蝎神经毒素，即昆虫毒素AaH IT1和AaH IT2。对AaH IT2进行了测序，发现它与之前唯一测序过的昆虫毒素AaH IT在四个氨基酸位置上存在差异[达尔邦，H.，兹洛特金，E.，科佩扬，C.，范·里特肖滕，J.，&罗沙特，H.（1982年）《国际肽与蛋白质研究杂志》20，320 - 330]，AaH IT对麻痹蝇幼虫具有同等潜力。AaH IT1与AaH IT相差一个氨基酸残基，对其碱性氨基酸残基进行了选择性化学修饰。对六种衍生物进行了表征。测定了它们对蝇幼虫和蟑螂的毒性，并测量了它们对来自蟑螂神经索的AaH IT1突触体受体的亲和力。组氨酸30、赖氨酸34和精氨酸60的修饰对生物活性没有显著影响。然而，赖氨酸28或赖氨酸51的修饰表明这两个氨基酸对毒性很重要。此外，赖氨酸28和赖氨酸51的同时修饰导致生物活性累积下降。AaH IT1和AaH IT2显示出相似的圆二色光谱。根据圆二色性数据估计了AaH IT2的二级结构含量。结果表明，这类毒素应具有一个额外的α螺旋区域和一条β折叠链，这在对哺乳动物有活性的毒素中未发现。试图在AaH IT2的氨基酸序列中定位这些二级结构特征表明，这两个区域将位于C末端最后20个氨基酸残基内。从这些关于二级结构的研究中，可以认为对昆虫有活性的毒素在结构上比那些对哺乳动物有活性的毒素更受限制；分子灵活性的降低可能至少部分地导致了这些毒素对昆虫钠通道所观察到的特异性。此外，在昆虫毒素中发现的两个α螺旋包围了两个保守的赖氨酸28和赖氨酸51，因此可能与昆虫毒素的毒性位点有关。
